Transcriptional activation of the HO-1 gene by lipopolysaccharide is mediated by 5' distal enhancers: role of reactive oxygen intermediates and AP-1.
Heme oxygenase-1 (HO-1) is a stress-response protein, the expression of which is transcriptionally regulated by agents that cause oxidative stress. We have previously shown that lipopolysaccharide (LPS)-induced HO-1 gene transcription in RAW 264.7 macrophage cells is mediated by a distal enhancer called SX2, located 4 kb upstream from the HO-1 transcription initiation site (Am. J. Respir. Cell Mol. Biol. 1995;13:387-398). We have recently identified a second distal enhancer, called AB1, located 6 kb upstream from the SX2 distal enhancer (J. Biol. Chem. 1995;270:11977-11984). Here we report the extension of our studies to investigate whether the AB1 distal enhancer and/or other potential regulatory elements in the entire 5' distal flanking sequences (11-kb region) of the HO-1 gene may also mediate HO-1 gene transcription in response to LPS. Using deletional analysis, we found that the AB1 enhancer also mediates LPS-induced HO-1 gene transcription. Mutational analysis of the AB1 enhancer and electrophoretic-mobility-shift assays of nuclear extracts from LPS-treated cells further demonstrated that the transcription factor activator protein-1 (AP-1) is critical for AB1-mediated HO-1 gene activation by LPS. We also found increased expression of AP-1 family members c-fos and c-jun by Northern blot analyses after treatment with LPS. Further, we observed that LPS-treated RAW 264.7 cells produced high levels of reactive oxygen intermediates (ROI) as measured through flow-cytometric analysis of dichlorofluoroscein (DCF)-stained cells. Treatment of cells with the antioxidants N-acetyl-L-cysteine (NAC) and dimethyl sulfoxide (DMSO) not only blunts LPS-induced production of ROI, but also significantly attenuates LPS-induced HO-1 messenger RNA (mRNA) expression and gene transcription. Taken together, these data suggest that LPS regulates HO-1 gene transcription in part by inducing the production of ROI, which initiate signal-transduction pathway(s) leading to the activation of AP-1-dependent HO-1 gene transcription.
Camhi
S L
SL
Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. scamhi@resp-sci.arizona.edu
Alam
J
J
Wiegand
G W
GW
Chin
B Y
BY
Choi
A M
AM
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002457:Cell Extracts; D002460:Cell Line; D002467:Cell Nucleus; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D016762:Genes, fos / Q000235:genetics; D016758:Genes, jun / Q000235:genetics; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics*; D051547:Heme Oxygenase-1; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages; D008565:Membrane Proteins; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis; D017382:Reactive Oxygen Species / Q000502:physiology*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D017384:Sequence Deletion; D018808:Transcription Factor AP-1 / Q000502:physiology; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics*
D000975:Antioxidants; D002457:Cell Extracts; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D018808:Transcription Factor AP-1; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse; D000111:Acetylcysteine; D004121:Dimethyl Sulfoxide